This research will study the intracellular transport and metabolism of liposomes that become cell-associated by endocytosis. Fluorescent and isotopically-labeled lipids will be prepared which have properties similar to lipids with normal fatty acids chains. Unlike most fluorescent lipids now available, these probes will not undergo appreciable rates of monomer transfer between bilayers ("lipid transfer"). Lipids probes will be incorporated into liposomes and incubated with mammalian cells in monolayer culture. Control experiments will be carried out to establish that most liposomal lipid becomes cell-associated by endocytosis, and not by lipid transfer, fusion, or absorption to the cell surface. After endocytosis, the intracellular metabolism and reutilization of lipid will be followed. Intracellular location will be determined by fluorescence microscopy and correlated with the conversion of fluorescent molecules to products of lipid biosynthesis. The metabolism of normal liposomes will be compared to the fate of pH-sensitive liposomes, which fuse with endosomal membranes prior to delivery to lysosomes. Cells will be treated with amphiphilic cations that are known to cause phospholipidosis (abnormal accumulation of lipid in lysosomes). The proposed experiments will evaluate the effect of these agents on 1) rate of uptake of liposomes, 2) rate of breakdown and release of lipid from endocytic compartments, 3) alterations in the reutilization of lysosomal products for lipid biosynthesis. It is a goal of this research to develop a convenient in vitro system for screening the effect of pharmacologic agents on lysosomal lipid metabolism.